P2X receptors are trimeric cation channels that open in response to binding of extracellular ATP. Each subunit contains a large extracellular ligand binding domain and two flanking transmembrane (TM) helices that form the pore, but the extent of gating motions of the TM helices is unclear. We probed these motions using ivermectin (IVM), a macrocyclic lactone that stabilizes the open state of P2X4 receptor channels. We find that IVM partitions into lipid membranes and that transfer of the TM regions of P2X4 receptors is sufficient to convey sensitivity to the lactone, suggesting that IVM interacts most favorably with the open conformation of the two TM helices at the protein-lipid interface. Scanning mutagenesis of the two TMs identifies residues that change environment between closed and open states, and substitutions at a subset of these positions weaken IVM binding. The emerging patterns points to widespread rearrangements of the TM helices during opening of P2X receptor channels.